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PubMed1994

Isolation of four major subunits from Clostridium thermocellum cellulosome and their synergism in the hydrolysis of crystalline cellulose.

The cellulosome of Clostridium thermocellum, purified by affinity chromatography, was dissociated under mild conditions and separated by SDS-PAGE. Two major p-nitrophenylcellobiosidases (PNPCases I and II) corresponding to the S5 (103 kDa) and S8 (78 kDa) subunits and one major carboxymethylcellulase (CMCase) coinciding with the S11 (60.5 kDa) subunit were isolated and characterized using carboxymethylcellulose (CMC), H3PO4-swollen cellulose and cello-oligosaccharides. Both PNPCases showed little effect on the viscosity of CMC and released twice as much total sugar as reducing sugar from H3PO4-swollen cellulose. The CMCase released ten times more total sugar than reducing sugar from H3PO4-swollen cellulose and reduced the viscosity of CMC rapidly. None of these enzymes was active on cellotriose. Both PNPCases released cellobiose from cellotetraose, and cellobiose and cellotriose from cellopentaose. In contrast, CMCase was active only on cellopentaose and released mainly glucose. Use of MeUmb(Glc)n revealed that both PNPCases cleaved preferentially either the second or fourth linkage from the non-reducing end while the CMCase was specific for the internal glycosidic bonds. Thus, the PNPCases and CMCase behaved as typical exo- and endoglucanases, respectively. When tested individually, all three enzymes degraded Avicel only to a small extent. A 1.5-2.0-fold increase in sugar release was observed when CMCase was combined with either PNPCase I, II or both. Combining S1 with either PNPCase II or CMCase resulted in fourfold synergism in the hydrolysis of Avicel. Synergism was sevenfold when all three enzymes were combined with S1.

Bacterial Proteins↗
PubMed1992

Identification of the cellulose-binding domain of the cellulosome subunit S1 from Clostridium thermocellum YS.

The 3' region of a gene designated cipB, which shows strong homology with cipA that encodes the cellulosome SL subunit of Clostridium thermocellum ATCC 27405, was isolated from a gene library of C. thermocellum strain YS. The truncated S1 protein encoded by the cipB derivative bound tightly to cellulose. The cellulose-binding domain in this polypeptide consisted of a C-terminal proximal 167 residue sequence which showed complete identity with residues 337-503 of mature SL from C. thermocellum strain ATCC 27405. The cellulose-binding domain interacted with both crystalline and amorphous cellulose, but not with xylan.

Amino Acid Sequence↗
PubMed2004

Cohesin-dockerin interaction in cellulosome assembly: a single Asp-to-Asn mutation disrupts high-affinity cohesin-dockerin binding.

The cohesive cellulosome complex is sustained by the high-affinity cohesin-dockerin interaction. In previous work, we demonstrated that a single Thr-to-Leu replacement in the Clostridium thermocellum dockerin component differentiates between non-recognition and high-affinity recognition by the interspecies rival cohesin from C. cellulolyticum. In this report, we show that a single Asp-to-Asn substitution on the cohesin counterpart also disrupts normal recognition of the dockerin. The Asp34 carboxyl group of the cohesin appears to play a central role in the resultant hydrogen-bonding network as an acceptor of two crucial hydrogen bonds from Ser45 of the dockerin domain. The results underscore the fragile nature of the intermolecular contact interactions that maintain this very high-affinity protein--protein interaction.

Amino Acid Sequence↗
PubMed2002

The crystal structure and catalytic mechanism of cellobiohydrolase CelS, the major enzymatic component of the Clostridium thermocellum Cellulosome.

Cellobiohydrolase CelS plays an important role in the cellulosome, an active cellulase system produced by the thermophilic anaerobe Clostridium thermocellum. The structures of the catalytic domain of CelS in complex with substrate (cellohexaose) and product (cellobiose) were determined at 2.5 and 2.4 A resolution, respectively. The protein folds into an (alpha/alpha)(6) barrel with a tunnel-shaped substrate-binding region. The conformation of the loops defining the tunnel is intrinsically stable in the absence of substrate, suggesting a model to account for the processive mode of action of family 48 cellobiohydrolases. Structural comparisons with other (alpha/alpha)(6) barrel glycosidases indicate that CelS and endoglucanase CelA, a sequence-unrelated family 8 glycosidase with a groove-shaped substrate-binding region, use the same catalytic machinery to hydrolyze the glycosidic linkage, despite a low sequence similarity and a different endo/exo mode of action. A remarkable feature of the mechanism is the absence, from CelS, of a carboxylic group acting as the base catalyst. The nearly identical arrangement of substrate and functionally important residues in the two active sites strongly suggests an evolutionary relationship between the cellobiohydrolase and endoglucanase families, which can therefore be classified into a new clan of glycoside hydrolases.

Catalytic Domain↗
PubMed2000

Isolation and properties of a cellulosome-type multienzyme complex of the thermophilic Bacteroides sp. strain P-1.

The extracellular form of cellulosome-type multienzyme complex of thermophilic Bacteroides sp. strain P-1 which was isolated from the anaerobic digester, is described. Multienzyme complex was isolated from the culture supernatant by an adsorption-desorption affinity chromatography on microcrystalline cellulose. The isolated multienzyme complex was found to form a complex that exhibited a high molecular weight (estimated at more than 1400 kDa) and was quite stable, requiring strong denaturing condition for dissociation. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate resolved multienzyme complex into at least 12 subunits with the molecular weight range of 49 to 209 kDa, respectively. The isolated multienzyme complex showed cellulose-binding ability, cellulase and xylanase activities and effected the hydrolysis of crystalline cellulose and lignocellulosic materials in the form of corncob, corn hull, rice straw, and sugarcane bagasse.

Journal Article↗
PubMed2003

Cel6A, a major exoglucanase from the cellulosome of the anaerobic fungi Piromyces sp. E2 and Piromyces equi.

Anaerobic fungi possess high cellulolytic activities, which are organised in high molecular mass (HMM) complexes. Besides catalytic modules, the cellulolytic enzyme components of these complexes contain non-catalytic modules, known as dockerins, that play a key role in complex assembly. Screening of a genomic and a cDNA library of two Piromyces species resulted in the isolation of two clones containing inserts of 5.5 kb (Piromyces sp. E2) and 1.5 kb (Piromyces equi). Both clones contained the complete coding region of a glycoside hydrolase (GH) from family 6, consisting of a 20 amino acid signal peptide, a 76 (sp. E2)/81 (P. equi) amino acid stretch comprising two fungal non-catalytic docking domains (NCDDs), a 24 (sp. E2)/16 (P. equi) amino acid linker, and a 369 amino acid catalytic module. Homology modelling of the catalytic module strongly suggests that the Piromyces enzymes will be processive cellobiohydrolases. The catalytic residues and all nearby residues are conserved. The reaction is thus expected to proceed via a classical single-displacement (inverting) mechanism that is characteristic of this family of GHs. The enzyme, defined as Cel6A, encoded by the full-length Piromyces E2 sequence was expressed in Escherichia coli. The recombinant protein expressed had a molecular mass of 55 kDa and showed activity against Avicel, supporting the observed relationship of the sequence to those of known cellobiohydrolases. Affinity-purified cellulosomes of Piromyces sp. E2 were analysed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) electrophoresis. A major band was detected with the molecular weight of Cel6A. A tryptic fingerprint of this protein confirmed its identity.

Amino Acid Sequence↗
PubMed1996

Expression, purification and crystallization of a cohesin domain from the cellulosome of Clostridium thermocellum.

The cellulosome of the cellulolytic bacterium, Clostridium thermocellum, is a multi-enzyme complex in which the enzymatic (cellulolytic) subunits are attached to a unique nonhydrolytic subunit called scaffoldin. The attachment is mediated by two mutually interacting domains: namely multiple cohesin domains on the scaffoldin subunit and a dockerin domain on each of the enzymatic subunits. Knowledge of the three-dimensional structure of each of the interacting components would be critical to a better understanding of the cohesin-dockerin interaction at the molecular level. In this report, we describe the purification of one of the nine cohesin domains of the scaffoldin subunit from C. thermocellum. A DNA segment containing the cohesin 2 sequence was fused to a hexa-histidine tag, and the resultant construct was expressed in Escherichia coli. The expressed peptide was efficiently isolated by metal-chelate affinity chromatography. The purified recombinant form of the cohesin was crystallized pending determination of its structure.

Amino Acid Sequence↗
PubMed1997

Synergism between the cellulosome-integrating protein CipA and endoglucanase CelD of Clostridium thermocellum.

The cellulosome-integrating protein CipA of Clostridium thermocellum was produced from a recombinant clone of Escherichia coli and purified by cellulose affinity and anion exchange chromatography. Two active forms of C. thermocellum endoglucanase CelD were tested for binding and hydrolytic activity on Avicel in the presence and in the absence of CipA. One was 68 kDa CelD, which contains an intact dockerin domain. The other was 65 kDa CelD, in which the dockerin domain is partially deleted. CipA promoted quantitative binding of 68 kDa CelD to Avicel and enhanced its Avicelase activity by at least ten-fold. By contrast, CipA had no effect on the activity nor on the cellulose-binding affinity of the truncated 65 kDa form. These results show that interaction between CipA and the catalytic component CelD is needed for the degradation of cellulose and confirm that the interaction is mediated by the dockerin domain of CelD.

Bacterial Proteins↗
PubMed1996

Characterization of engF, a gene for a non-cellulosomal Clostridium cellulovorans endoglucanase.

A new Clostridium cellulovorans (strain ATCC 35296) endoglucanase gene engF has been isolated and sequenced. The gene contains 1671 bp and codes for a protein containing 557 amino acids and a mass of 60.1 kDa. A putative signal peptide of 29 amino acids is present and the mature protein has a mass of 57.1 kDa. EngF does not have amino acid sequence homology to previously isolated EngB and EngD, but does show sequence homology to family 5 glycosyl hydrolases from Bacillus, Erwinia carotovora, and C. acetobutylicum species. EngF is not a component of the cellulosome and does not contain a duplicated sequence (DS) at its C-terminal region. EngF is capable of binding to cellulose and hydrolyzing carboxymethylcellulose but not xylan. The cellulose binding domain (CBD) differs from types I, II and III CBDs and no obvious homology has been found to other CBD types. The maximum activity of EngF occurs at pH 5.5 and at 47 degrees C. Its properties suggest that EngF plays an ancillary role in the degradation of cellulosic materials.

Amino Acid Sequence↗
PubMed1998

Cellulose, cellulases and cellulosomes.

The structural complexity and rigidity of cellulosic substrates have given rise to a phenomenal diversity of degradative enzymes--the cellulases. Cellulolytic microorganisms produce a wide variety of different catalytic and noncatalytic enzyme modules, which form the cellulases and act synergistically on their substrate. In some microbes, several types of cellulases are organized into an elaborate multifunctional supramolecular complex, known as the cellulosome. A combination of molecular genetic, biochemical, chemical, crystallographic and microscopic techniques are paving the way for new insights into both the structure of cellulose and the mechanisms of its hydrolysis.

Bacteria↗
PubMed2001

Structural basis for the substrate specificity of the feruloyl esterase domain of the cellulosomal xylanase Z from Clostridium thermocellum.

Feruloyl esterases function in the cleavage of ferulic acid's bonds to arabinoxylan and pectin where the ferulic acid moieties cross-link the layers of polysaccharide chains within hemicellulose. This work presents the crystal structure of FAE_XynZ, the domain of Clostridium thermocellum's cellulosomal xylanase Z that displays feruloyl esterase activity. The structure was obtained via multiple isomorphous replacement with anomalous scattering (MIRAS) using three heavy atom derivatives and refined against X-ray diffraction data of up to 1.75 A resolution. The R-value of the final model was 0.187 (R(free) = 0.21). FAE_XynZ displays an eight-stranded alpha/beta-fold with the characteristic "catalytic triad" at the heart of the active site. To define the substrate specificity determinants of the enzyme, the crystal structures of FAE_XynZ and the inactive FAE_XynZ(S172A) mutant were determined in complexes with the feruloyl-arabinoxylans FAXX and FAX(3), respectively. In the complex crystals, the ferulic acid moieties are clearly recognizable and allowed identification of the hydrophobic binding pocket. The carbohydrate part of both substrates is not visible in either structure. The location of the putative carbohydrate binding-pocket was inferred based on the location and orientation of the adjacent ferulic acid molecule. Five of the six residues lining the pocket were found to be conserved in FAE A from Orpinomyces sp., which further supports the proposed role of these amino acids.

Alanine↗
PubMed1999

Cellulosome from Clostridium cellulolyticum: molecular study of the Dockerin/Cohesin interaction.

Clostridium cellulolyticum produces cellulolytic complexes (cellulosomes) made of 10-13 cell wall degrading enzymes tightly bound to a scaffolding protein (CipC) by means of their dockerin domain. It has previously been shown that the receptor domains in CipC are the cohesin domains and that the cohesin/dockerin interaction is calcium-dependent. In the present study, surface plasmon resonance was used to demonstrate that the free cohesin1 from CipC and dockerin from CelA have the same K(D) (2.5 x 10(-)(10) M) as that of the entire CelA and a larger fragment of CipC, the latter of which contains, in addition to cohesin1, a cellulose binding domain and a hydrophilic domain of unknown function. This demonstrates that neither the catalytic domain of CelA nor the noncohesin domains of CipC have any influence on the interaction. Dockerin domains are composed of two conserved segments of 22 residues: removal of the second segment abolishes the affinity for cohesin1, whereas modified dockerins having twice the first segment, twice the second, or both segments but in a reverse order have K(D) values for cohesin1 in the same range as that observed for wild-type dockerin. These data indicate that if two segments are required for the complexation with the cohesin, segments 1 and 2 are similar enough to replace each other. Calcium overlay experiments revealed that the dockerin domain has one calcium binding site per conserved segment. Circular dichroism performed on wild-type and mutant dockerins indicates that this domain is well structured and that removal of calcium only weakly affects the secondary structure, which remains 40-45% helical.

Amino Acid Sequence↗
PubMed2001

Characterization of a cellulosome dockerin domain from the anaerobic fungus Piromyces equi.

The recycling of photosynthetically fixed carbon in plant cell walls is a key microbial process. In anaerobes, the degradation is carried out by a high molecular weight multifunctional complex termed the cellulosome. This consists of a number of independent enzyme components, each of which contains a conserved dockerin domain, which functions to bind the enzyme to a cohesin domain within the protein scaffoldin protein. Here we describe the first three-dimensional structure of a fungal dockerin, the N-terminal dockerin of Cel45A from the anaerobic fungus Piromyces equi. The structure contains a novel fold of 42 residues. The ligand binding site consists of residues Trp 35, Tyr 8 and Asp 23, which are conserved in all fungal dockerins. The binding site is on the opposite side of the N- and C-termini of the molecule, implying that tandem dockerin domains, seen in the majority of anaerobic fungal plant cell wall degrading enzymes, could present multiple simultaneous binding sites and, therefore, permit tailoring of binding to catalytic demands.

Amino Acid Sequence↗
PubMed1997

Interaction between Clostridium thermocellum endoglucanase CelD and polypeptides derived from the cellulosome-integrating protein CipA: stoichiometry and cellulolytic activity of the complexes.

Four mini-scaffoldins were constructed from modules derived from the Clostridium thermocellum cellulosome-integrating protein CipA. Cip7 and Cip6 contained one and two cohesin modules respectively. Cip14 and Cip16, also containing one and two cohesin modules respectively, were flanked by a cellulose-binding domain. Endoglucanase CelD formed stable complexes with all mini-scaffoldins. Analytical ultracentrifugation of the complexes showed that 1 mol of CelD bound per mol of Cip14, and 2 mol of CelD bound per mol of Cip16. Under the conditions used for assaying cellulase activity, 96% of CelD alone bound to Avicel. Association with Cip14 or Cip16 increased the cellulose binding of CelD to 99%, while association with Cip7 or Cip6 decreased binding to 79 and 75% respectively. The hydrolytic activity of CelD against Avicel was increased 3-fold in complexes with Cip14 and Cip16, but remained substantially the same in complexes with Cip6 and Cip7. Addition of whole CipA also enhanced the efficiency of Avicel hydrolysis by CelD. However, even at an optimal ratio of the components, CelD-CipA complexes were somewhat less active than complexes of CelD with Cip14 or Cip16. These results suggest that the synergism observed between CelD and Cip14 or Cip16 is mostly due to the presence of the cellulose-binding domain, which promotes productive binding of the enzyme.

Adsorption↗
PubMed2002

A newly described cellulosomal cellobiohydrolase, CelO, from Clostridium thermocellum: investigation of the exo-mode of hydrolysis, and binding capacity to crystalline cellulose.

The sequence of the celO gene from Clostridium thermocellum F7 was determined. The gene product, cellulase CelO (Ct-Cel5F), had a modular structure consisting of a carbohydrate-binding module of the CBM3 family and a catalytic domain of the glycosyl hydrolase family 5. The presence of the dockerin module indicated that the enzyme was a component of the cellulosome complex. The thermostable recombinant gene product was active on cellodextrins, barley beta-glucan, carboxymethylcellulose and insoluble cellulose. Cellobiose was the only product released from amorphic and crystalline cellulose, cellotetraose and higher cello-oligosaccharides, identifying CelO as a cellobiohydrolase. The cleavage pattern of p-nitrophenyl beta-D-cellotetraoside, blockage of the hydrolysis of NaBH(4)-reduced cellopentaose and the reduction in substrate viscosity suggested activity from the reducing end in a processive mode after making random cuts. Binding to insoluble, i.e. amorphous, and crystalline cellulose was mediated by the carbohydrate-binding module CBM3b, with a preference for the crystalline substrate.

Cellobiose↗
PubMed2000

Crystallization and preliminary X-ray analysis of the Clostridium thermocellum cellulosome xylanase Z feruloyl esterase domain.

Feruloyl esterases cleave ferulic acid from arabinoxylan and pectin. Feruloyl groups are believed to crosslink the polysaccharide chain within the polymer and to link hemicellulose to lignin, which may play a role in controlling the growth of plants. The Clostridium thermocellum cellulosome xylanase Z feruloyl esterase was expressed in Escherichia coli, purified and crystallized. The crystals diffract to 2.4 A resolution and belong to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 43.14, b = 63.77, c = 79.57 A. Assuming one molecule per asymmetric unit, the Matthews coefficient is calculated to be 1.87 A(3) Da(-1), which corresponds to a solvent content of 34%.

Carbohydrate Sequence↗
PubMed2000

Structure of a family IIIa scaffoldin CBD from the cellulosome of Clostridium cellulolyticum at 2.2 A resolution.

The crystal structure of the family IIIa cellulose-binding domain (CBD) from the cellulosomal scaffoldin subunit (CipC) of Clostridium cellulolyticum has been determined. The structure reveals a nine-stranded jelly-roll topology which exhibits distinctive structural elements consistent with family III CBDs that bind crystalline cellulose. These include a well conserved calcium-binding site, a putative cellulose-binding surface and a conserved shallow groove of unknown function. The CipC CBD structure is very similar to the previously elucidated family IIIa CBD from the CipA scaffoldin of C. thermocellum, with some minor differences. The CipC CBD structure was also compared with other previously described CBD structures from families IIIc and IV derived from the endoglucanases of Thermomonospora fusca and Cellulomonas fimi, respectively. The possible functional consequences of structural similarities and differences in the shallow groove and cellulose-binding faces among various CBD families and subfamilies are discussed.

Amino Acid Sequence↗
PubMed1997

Crystallization and preliminary X-ray analysis of a cohesin domain of the cellulosome from Clostridium thermocellum.

Recombinant cohesin-2, a unique type of protein-recognition domain from the cellulosome of Clostridium thermocellum, has been crystallized by the hanging-drop vapor-diffusion method. The crystals are monoclinic, space group C2 with unit-cell dimensions a = 79.91, b = 47.86, c = 51.13 A, beta = 126.77 degrees. There is most likely to be one molecule per asymmetric unit, corresponding to a packing density of 2.16 A(3) Da(-1). The crystals diffract to beyond 2.3 A on a conventional laboratory rotating-anode source.

Journal Article↗